Mutational analysis of the control cable that mediates transmembrane signaling in the Escherichia coli serine chemoreceptor

During transmembrane signaling by Escherichia coli Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment.     

HAMP domain structural determinants for signalling and sensory adaptation in Tsr,the Escherichia coli serine chemoreceptor

HAMP domains mediate input–output transactions in many bacterial signalling proteins. To clarify the mechanistic logic of HAMP signalling, we constructed Tsr‐HAMP deletion derivatives and characterized their steady‐state signal outputs and sensory adaptation properties with flagellar rotation and receptor methylation assays. Tsr molecules lacking the entire HAMP domain or just the HAMP‐AS2 helix generated clockwise output signals, confirming that kinase activation is the default output state of the chemoreceptor signalling domain and that attractant stimuli shift HAMP to an overriding kinase‐off signalling state to elicit counter‐clockwise flagellar responses. Receptors with deletions of the AS1 helices, which free the AS2 helices from bundle‐packing constraints, exhibited kinase‐off signalling behaviour that depended on three C‐terminal hydrophobic residues of AS2. We conclude that AS2/AS2′ packing interactions alone can play an important role in controlling output kinase activity. Neither kinase‐on nor kinase‐off HAMP deletion outputs responded to sensory adaptation control, implying that out‐of‐range conformations or bundle‐packing stabilities of their methylation helices prevent substrate recognition by the adaptation enzymes. These observations support the previously proposed biphasic, dynamic‐bundle mechanism of HAMP signalling and additionally show that the structural interplay of helix‐packing interactions between HAMP and the adjoining methylation helices is critical for sensory adaptation control of receptor output.     

Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli.

The chemoreceptors responsible for the repellent response of Escherichia coli to phenol were investigated. In the absence of all four known methyl-accepting chemoreceptors (Tar, Tsr, Trg, and Tap), cells showed no response to phenol. However, when Trg, which mediates the attractant response to ribose and galactose, was introduced via a plasmid, the cells acquired a repellent response to phenol. About 1 mM phenol induced a clear repellent response; this response was suppressed by 1 mM ribose. Thus, Trg mediates the repellent response to phenol. Mutant Trg proteins with altered sensing for ribose and galactose showed a normal response to phenol, indicating that the interaction site for phenol differs from that for the ribose- and galactose-binding proteins. Tap, which mediates the attractant response to dipeptides, mediated a weaker repellent response to phenol. Tsr, which mediates the attractant response to serine, mediated an even weaker response to phenol. Trg and Tap were also found to function as intracellular pH sensors. Upon a pH decrease, Trg mediated an attractant response, whereas Tap mediated a repellent response. These results indicate that all the receptors in E. coli have dual functions, mediating both attractant and repellent responses.     

Sensing of cytoplasmic pH by bacterial chemoreceptors involves the linker region that connects the membrane-spanning and the signal-modulating helices.

The two major chemoreceptors of Escherichia coli, Tsr and Tar, mediate opposite responses to the same changes in cytoplasmic pH (pH(i)). We set out to identify residues involved in pH(i) sensing to gain insight into the general mechanisms of signaling employed by the chemoreceptors. Characterization of various chimeras of Tsr and Tar localized the pH(i)-sensing region to Arg(259)-His(267) of Tar and Gly(261)-Asp(269) of Tsr. This region of Tar contains three charged residues (Arg(259)-Ser(261), Asp(263), and His(267)) that have counterparts of opposite charge in Tsr (Gly(261)-Glu(262), Arg(265), and Asp(269)). The replacement of all of the three charged residues in Tar or Arg(259)-Ser(260) alone by the corresponding residues of Tsr reversed the polarity of pH(i) response, whereas the replacement of Asp(263) or His(267) did not change the polarity but altered the time course of pH(i) response. These results suggest that the electrostatic properties of a short cytoplasmic region within the linker region that connects the second transmembrane helix to the first methylation helix is critical for switching the signaling state of the chemoreceptors during pH sensing. Similar conformational changes of this region in response to external ligands may be critical components of transmembrane signaling.     

Transmembrane signaling of chemotaxis receptor tar: insights from molecular dynamics simulation studies

Transmembrane signaling of chemotaxis receptors has long been studied, but how the conformational change induced by ligand binding is transmitted across the bilayer membrane is still elusive at the molecular level. To tackle this problem, we carried out a total of 600-ns comparative molecular dynamics simulations (including model-building simulations) of the chemotaxis aspartate receptor Tar (a part of the periplasmic domain/transmembrane domain/HAMP domain) in explicit lipid bilayers. These simulations reveal valuable insights into the mechanistic picture of Tar transmembrane signaling. The piston-like movement of a transmembrane helix induced by ligand binding on the periplasmic side is transformed into a combination of both longitudinal and transversal movements of the helix on the cytoplasmic side as a result of different protein-lipid interactions in the ligand-off and ligand-on states of the receptor. This conformational change alters the dynamics and conformation of the HAMP domain, which is presumably a mechanism to deliver the signal from the transmembrane domain to the cytoplasmic domain. The current results are consistent with the previously suggested dynamic bundle model in which the HAMP dynamics change is a key to the signaling. The simulations provide further insights into the conformational changes relevant to the HAMP dynamics changes in atomic detail.     

Mutational analyses of HAMP helices suggest a dynamic bundle model of input–output signalling in chemoreceptors

To test the gearbox model of HAMP signalling in the Escherichia coli serine receptor, Tsr, we generated a series of amino acid replacements at each residue of the AS1 and AS2 helices. The residues most critical for Tsr function defined hydrophobic packing faces consistent with a four-helix bundle. Suppression patterns of helix lesions conformed to the predicted packing layers in the bundle. Although the properties and patterns of most AS1 and AS2 lesions were consistent with both proposed gearbox structures, some mutational features specifically indicate the functional importance of an x-da bundle over an alternative a-d bundle. These genetic data suggest that HAMP signalling could simply involve changes in the stability of its x-da bundle. We propose that Tsr HAMP controls output signals by modulating destabilizing phase clashes between the AS2 helices and the adjoining kinase control helices. Our model further proposes that chemoeffectors regulate HAMP bundle stability through a control cable connection between the transmembrane segments and AS1 helices. Attractant stimuli, which cause inward piston displacements in chemoreceptors, should reduce cable tension, thereby stabilizing the HAMP bundle. This study shows how transmembrane signalling and HAMP input–output control could occur without the helix rotations central to the gearbox model.     

Role of threonine residue 154 in ligand recognition of the tar chemoreceptor in Escherichia coli.

The Tar chemoreceptor of Escherichia coli mediates attractant responses to aspartate, maltose, and phenol, repellent responses to Ni2+ and Co2+, and thermoresponses. To understand the role of threonine residue 154, which is located in the ligand-binding domain of Tar, we replaced the residue with serine, isoleucine, and proline by site-directed mutagenesis. The replacements caused reductions in aspartate sensing but had only a small effect on maltose sensing and almost no effect on phenol sensing, repellent sensing, and thermosensing. These results indicate that Thr-154 of Tar is rather specifically involved in aspartate sensing. The reductions in the response threshold for aspartate by the replacements with serine, isoleucine, and proline were less than 1, about 2, and more than 5 orders of magnitude, respectively. When the corresponding threonine residue in the Tsr chemoreceptor was replaced with the same amino acids, roughly similar reductions in the response threshold for serine resulted. Thus, these threonine residues seem to have a common role in detecting the aspartate and serine attractant families. A mechanism by which these chemoreceptors detect the amino acid attractants is discussed.     

Modeling the transmembrane domain of bacterial chemoreceptors

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.     

Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.     

Mutational analysis of the connector segment in the HAMP domain of Tsr, the Escherichia coli serine chemoreceptor

HAMP domains are approximately 50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemoreceptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic responses. We created random amino acid replacements at each of the 14 connector residues of Tsr-HAMP to identify those that are critical for Tsr function. In all, we surveyed 179 connector missense mutants and identified three critical residues (G235, L237, and I241) at which most replacements destroyed Tsr function and another important residue (G245) at which most replacements impaired Tsr function. The region surrounding G245 tolerated 1-residue deletions and insertions of up to 10 glycines, suggesting a role as a relatively nonspecific, flexible linker. The critical connector residues are consistent with a structural model of the Tsr-HAMP domain based on the solution structure of an isolated thermophile HAMP domain (M. Hulko, F. Berndt, M. Gruber, J. U. Linder, V. Truffault, A. Schultz, J. Martin, J. E. Schultz, A. N. Lupas, and M. Coles, Cell 126:929-940, 2006) in which G235 defines a critical turn at the C terminus of the first helix and L237 and I241 pack against the helices, perhaps to stabilize alternative HAMP signaling conformations. Most I241 lesions locked Tsr signal output in the kinase-on mode, implying that this residue is responsible mainly for stabilizing the kinase-off signaling state. In contrast, lesions at L237 resulted in a variety of aberrant output patterns, suggesting a role in toggling output between signaling states.     

Mutationally altered signal output in the Nart (NarX-Tar) hybrid chemoreceptor

Signal-transducing proteins that span the cytoplasmic membrane transmit information about the environment to the interior of the cell. In bacteria, these signal transducers include sensor kinases, which typically control gene expression via response regulators, and methyl-accepting chemoreceptor proteins, which control flagellar rotation via the CheA kinase and CheY response regulator. We previously reported that a chimeric protein (Nart) that joins the ligand-binding, transmembrane, and linker regions of the NarX sensor kinase to the signaling and adaptation domains of the Tar chemoreceptor elicits a repellent response to nitrate and nitrite. As with NarX, nitrate evokes a stronger response than nitrite. Here we show that mutations targeting a highly conserved sequence (the P box) in the periplasmic domain alter chemoreception by Nart and signaling by NarX similarly. In particular, the G51R substitution converts Nart from a repellent receptor into an attractant receptor for nitrate. Our results underscore the conclusion that the fundamental mechanism of transmembrane signaling is conserved between homodimeric sensor kinases and chemoreceptors. They also highlight the plasticity of the coupling between ligand binding and signal output in these systems.     

Functional Suppression of HAMP Domain Signaling Defects in the E. coli Serine Chemoreceptor

HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.     

Tryptophan residues flanking the second transmembrane helix (TM2) set the signaling state of the Tar chemoreceptor

The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster in patches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellar rotation. The functional domains of a receptor are physically separated by the cell membrane. Chemoeffectors bind to the extracellular (periplasmic) domain, and the cytoplasmic domain mediates signaling and adaptation. These two domains communicate through the second transmembrane helix (TM2) that connects them. In the high-abundance receptors Tar and Tsr, TM2 is flanked by tryptophan residues, which should localize preferentially to the interfacial zone between the polar and hydrophobic layers of the phospholipid bilayer. To investigate the functional significance of the Trp residues that flank TM2 of Tar, we used site-directed mutagenesis to generate the W192A and W209A substitutions. The W192A protein retains full activity in vivo and in vitro, but it increases the K(i) for aspartate in the in vitro assay 3-fold. The W209A replacement eliminates receptor-mediated stimulation of CheA in vitro, and it leads to an increased level of adaptive methylation in vivo. This phenotype in some respects mimics the changes seen upon binding aspartate. Since the W209A substitution may cause the C-terminus of TM2 to protrude farther into the cytoplasm, these results reinforce the hypothesis that aspartate binding causes a similar displacement. Moving Trp to each position from residue 206 to residue 212 generated a wide variety of Tar signaling states that are generally consistent with the predictions of the piston model of transmembrane signaling. None of these receptors was completely locked in one signaling mode, although most showed pronounced signaling biases. Our findings suggest that the Trp residues flanking TM2, especially Trp-209, are important in setting the baseline activity and ligand sensitivity of the Tar receptor. We also conclude that the Tyr-210 residue plays at least an auxiliary role in this control.     

Biphasic control logic of HAMP domain signalling in the Escherichia coli serine chemoreceptor

HAMP domains mediate input-output communication in many bacterial signalling proteins. To explore the dynamic bundle model of HAMP signalling (Zhou et al., Mol. Microbiol. 73: 801, 2009), we characterized the signal outputs of 118 HAMP missense mutants of the serine chemoreceptor, Tsr, by flagellar rotation patterns. Receptors with proline or charged amino acid replacements at critical hydrophobic packing residues in the AS1 and AS2 HAMP helices had locked kinase-off outputs, indicating that drastic destabilization of the Tsr-HAMP bundle prevents kinase activation, both in the absence and presence of the sensory adaptation enzymes, CheB and CheR. Attractant-mimic lesions that enhance the structural stability of the HAMP bundle also suppressed kinase activity, demonstrating that Tsr-HAMP has two kinase-off output states at opposite extremes of its stability range. HAMP mutants with locked-on kinase outputs appeared to have intermediate bundle stabilities, implying a biphasic relationship between HAMP stability and kinase activity. Some Tsr-HAMP mutant receptors exhibited reversed output responses to CheB and CheR action that are readily explained by a biphasic control logic. The findings of this study provide strong support for a three-state dynamic bundle model of HAMP signalling in Tsr, and possibly in other bacterial transducers as well.     

Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.     

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.     

Inversion of thermosensing property of the bacterial receptor Tar by mutations in the second transmembrane region

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar.     

Structure of the conserved HAMP domain in an intact, membrane-bound chemoreceptor: a disulfide mapping study

The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Recently, a structure was determined for a HAMP domain isolated from an unusual archeal membrane protein of unknown function [Hulko, M., et al. (2006) Cell 126, 929-940]. This study uses cysteine and disulfide chemistry to test this archeal HAMP model in the full-length, membrane-bound aspartate receptor of bacterial chemotaxis. The chemical reactivities of engineered Cys residues scanned throughout the aspartate receptor HAMP region are highly correlated with the degrees of solvent exposure of corresponding positions in the archeal HAMP structure. Both domains are homodimeric, and the individual subunits of both domains share the same helix-connector-helix organization with the same helical packing faces. Moreover, disulfide mapping reveals that the four helices of the aspartate receptor HAMP domain are arranged in the same parallel, four-helix bundle architecture observed in the archeal HAMP structure. One detectable difference is the packing of the extended connector between helices, which is not conserved. Finally, activity studies of the aspartate receptor indicate that contacts between HAMP helices 1 and 2' at the subunit interface play a critical role in modulating receptor on-off switching. Disulfide bonds linking this interface trap the receptor in its kinase-activating on-state, or its kinase inactivating off-state, depending on their location. Overall, the evidence suggests that the archeal HAMP structure accurately depicts the architecture of the conserved HAMP motif in transmembrane chemoreceptors. Both the on- and off-states of the aspartate receptor HAMP domain closely resemble the archeal HAMP structure, and only a small structural rearrangement occurs upon on-off switching. A model incorporating HAMP into the full receptor structure is proposed.     

Tuning a bacterial chemoreceptor with protein-membrane interactions

Chemoreceptors in Escherichia coli are homodimeric transmembrane proteins that convert environmental stimuli into intracellular signals controlling flagellar motion. Chemoeffectors bind to the extracellular (periplasmic) domain of the receptors, whereas their cytoplasmic domain mediates signaling and adaptation. The second transmembrane helix (TM2) connects these two domains. TM2 contains an aliphatic core flanked by amphipathic aromatic residues that have specific affinity for polar-hydrophobic membrane interfaces. We previously showed that Trp-209, near the cytoplasmic end of TM2, helps maintain the normal baseline-signaling state of the aspartate chemoreceptor (Tar) and that Tyr-210 plays an auxiliary role in this control. We have now repositioned the Trp-209/Tyr-210 pair in single-residue increments about the cytoplasmic polar-hydrophobic interface. Changes from WY-2 to WY+1 modulate the baseline-signaling state of the receptor in predictable and incremental steps that can be compensated by adaptive methylation/demethylation. Greater displacements, as in WY-3, WY+2, and WY+3, bias the receptor to the off kinase-inhibiting state or the on kinase-stimulating state, respectively, to a degree that cannot be fully compensated by the adaptation system. Aromatic residues analogous to Trp-209/Tyr-210 are present in other chemoreceptors and many transmembrane sensor kinases, where they may serve a similar function.     

Not too loose, not too tight--just right. Biphasic control of the Tsr HAMP domain

HAMP domains communicate between input and output signalling modules in a wide variety of bacterial sensor proteins. In the Tsr chemoreceptor, they convert a signal initiated by binding of serine to the periplasmic domain of the protein into regulation of receptor control of the CheA kinase, and ultimately of the direction of flagellar rotation. In this issue, Zhou et al. report an extensive mutational analysis of the Tsr HAMP domain that shows that it can assume a number of different signalling states, which presumably correspond to a variety of different conformations. The two conformational extremes of a tightly packed and a loosely packed HAMP four‐helix bundle support only low levels of CheA activity. Thus, Tsr HAMP does not function as a simple on‐off, two‐state device but rather as a dynamic structure with biphasic control. The normal physiological operating range of Tsr is proposed to be at intermediate degrees of packing of the HAMP four‐helix bundle, but HAMP domains in other proteins could occupy different portions of the conformational spectrum.